Integrated targeted metabolomics and complementary [α-15N] glutamine tracing reveal glutamine metabolic remodeling associated with RA-ILD

An integrated multi-level analytical framework was developed to characterize glutamine metabolic remodeling associated with RA-ILD. • Multi-matrix metabolomics revealed compartment-specific redistribution of glutamine-related amino acids. • [α- 15 N] glutamine tracing in vitro identified intracellular fibrotic redirection toward serine/glycine biosynthesis. • CTSs modulated intestinal transporters and suppressed the ATF4-associated serine/glycine program. Background Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a severe complication with limited therapeutic options, characterized by destructive joint lesions and pulmonary fibrosis involving excessive collagen deposition. Glutamine metabolism supplies precursors for collagen-related amino acid biosynthesis, which drives the fibrotic remodeling process, but its cross-organ redistribution and pathway-specific utilization remain difficult to resolve using conventional metabolomic readouts. An analytical strategy capable of integrating quantitative multi-matrix profiling with isotope-resolved nitrogen tracing is therefore needed to characterize alterations in glutamine-related amino acid metabolism associated with RA-ILD and its pharmacological modulation. Results We established a complementary analytical framework in which 3-trimethylsilyl-1-propanaminium chloride (TSPC) derivatization-assisted UPLC-QqQ-MS/MS targeted metabolomics and intestinal amino acid transporter profiling were applied to an RA-ILD rat model, while [α- 15 N] glutamine tracing was independently performed in a TGF-β1-induced fibrotic cell model to examine pathway-specific glutamine nitrogen utilization. The validated method enabled sensitive quantification of glutamine-related amino acids in serum, lung tissue, and cells. In RA-ILD rats, cross-matrix profiling revealed a compartment-dependent disturbance of glutamine-related amino acid distribution, characterized by pulmonary accumulation coupled with altered circulating availability. Clematis triterpenoid saponins (CTSs) partially normalized this metabolic imbalance and were associated with coordinated regulation of intestinal glutamine transporters. Stable-isotope tracing in a fibrotic cell model further showed preferential incorporation of glutamine-derived nitrogen into the serine-glycine axis, which was supported by increased expression of ATF4-associated serine-glycine biosynthetic enzymes in lung tissue. CTSs treatment attenuated the corresponding lung transcriptional alterations. Significance Together, in vivo multi-matrix metabolomics characterized systemic glutamine-related metabolic redistribution and its response to CTSs in RA-ILD, whereas complementary in vitro isotope tracing provided pathway-level evidence for preferential glutamine-derived nitrogen utilization through the serine-glycine axis under fibrotic stimulation. Such a complementary framework also provides a transferable analytical strategy for investigating cross-organ diseases and multi-level interventions of traditional Chinese medicines. Download: Download high-res image (371KB) Download: Download full-size image